Green synthesized zinc oxide nanoparticles mediated by Dysphania botrys extract: Structural characterization and biological applications

Abstract

Zinc oxide nanoparticles (ZnONPs) were prepared using Dysphania botrys extract. Successful NPs formation was confirmed by UV-Vis spectroscopy with a characteristic absorbance peak at 365 nm. XRD analysis revealed a hexagonal wurtzite structure with an average crystallite size of 9.96 nm, while FTIR spectra indicated the involvement of plant phytochemicals in nanoparticle stabilization. SEM images showed predominantly hexagonal morphology, and EDX analysis confirmed high purity, with zinc and oxygen as the major elements. GC–MS profiling of the plant extract identified 26 bioactive compounds, with humulane-1,6-dien-3-ol (29.4%) as the most abundant. The biosynthesized ZnONPs exhibited pronounced antibacterial activity against both Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Brine shrimp assays revealed concentration-dependent toxicity (100% mortality at ≥160 µg/mL), indicating that the biosynthesized ZnONPs had notable general cytotoxic potential, which warrants careful evaluation of environmental and biomedical safety. Meanwhile, DPPH assays revealed concentration-dependent antioxidant activity (58.8% at 200 µg/mL). Green synthesis using plant extracts has been proposed as a more environmentally benign approach and can reduce the use of hazardous reagents, although the resulting nanoparticles may still exhibit toxicity depending on their dose and properties.

Full Article

Green Synthesized Zinc Oxide Nanoparticles Mediated by Dysphania botrys Extract: Structural Characterization and Biological Applications

Abdul Nasir  ^a† Muhammad Tayyab  ,^{a †} Syed Jehangir Shah,^a Muhammad Sayyar Khan  ,^a Heba I. Mohamed  ,^b,* Haiam O. Elkatry  ,^c Marwa Ezz El-Din Ibrahim  ,^c Hossam S. El-Beltagi  ,^d Mohamed El Oirdi  ,^e and Abdelrahman R. Ahmed  ,^c,*

Zinc oxide nanoparticles (ZnONPs) were prepared using Dysphania botrys extract. Successful NPs formation was confirmed by UV-Vis spectroscopy with a characteristic absorbance peak at 365 nm. XRD analysis revealed a hexagonal wurtzite structure with an average crystallite size of 9.96 nm, while FTIR spectra indicated the involvement of plant phytochemicals in nanoparticle stabilization. SEM images showed predominantly hexagonal morphology, and EDX analysis confirmed high purity, with zinc and oxygen as the major elements. GC–MS profiling of the plant extract identified 26 bioactive compounds, with humulane-1,6-dien-3-ol (29.4%) as the most abundant. The biosynthesized ZnONPs exhibited pronounced antibacterial activity against both Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Brine shrimp assays revealed concentration-dependent toxicity (100% mortality at ≥160 µg/mL), indicating that the biosynthesized ZnONPs had notable general cytotoxic potential, which warrants careful evaluation of environmental and biomedical safety. Meanwhile, DPPH assays revealed concentration-dependent antioxidant activity (58.8% at 200 µg/mL). Green synthesis using plant extracts has been proposed as a more environmentally benign approach and can reduce the use of hazardous reagents, although the resulting nanoparticles may still exhibit toxicity depending on their dose and properties.

DOI: 10.15376/biores.21.2.5106-5121

Keywords: Antioxidant activity; Antimicrobial activity; Characterization; Cytotoxic assay; Gas chromatography–mass spectrometry

Contact information: a: Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, 25130 Peshawar, Pakistan; b: Department of Biological and Geological Sciences, Faculty of Education, Ain Shams University, Cairo, Egypt; c: Food and Nutrition Science Department, Agricultural Science and Food, King Faisal University, Al Ahsa 31982, Saudi Arabia; d: Agricultural Biotechnology Department, College of Agricultural and Food Science, King Faisal University, Al-Ahsa, 31982, Saudi Arabia; e: Department of Life Sciences, College of Science, King Faisal University, Al-Ahsa, 31982, Saudi Arabia; Corresponding authors: Hebaibrahim79@gmail.com; arahmed@kfu.edu.sa

†These authors contributed equally to this work.

Graphical Abstract

INTRODUCTION

Green synthesis of zinc oxide nanoparticles (ZnONPs) using plant extracts has emerged as an eco-friendly alternative to conventional physical and chemical routes, which often involve high energy inputs and/or toxic precursors and solvents, which may raise ecological and occupational safety concerns compared with plant-mediated approaches (Al-Rajhi et al. 2022; Abdelghany et al. 2023; Al-darwesh et al. 2024). Usually, nanoparticle size is between 1 nm and 100 nm, exhibiting distinctive features such as a large surface area-to-volume ratio and quantum effects, which contribute to their enhanced reactivity and multifunctionality (Ahmed et al. 2025).

ZnONPs are typically synthesized via chemical vapor deposition, sol-gel methods, hydrothermal methods, or co-precipitation methods. Although such approaches can produce excellent nanoparticles, they often involve the use of toxic chemicals and extreme conditions, which can have ecological potential risks (Jha et al. 2025). Green synthesis processes, prepared by bottom-up approaches and considered environmentally friendly, have sparked renewed interest in nanoparticle manufacturing in recent decades. This approach produces nanoparticles with a host of natural resources, such as bacteria and plants (Rashid et al. 2024). ZnONPs are commonly synthesized using plants or their parts, such as bark, stem, leaves, fruit, and seeds (Abdelhady et al. 2024; Alam et al. 2025). In these methods, plant phytochemicals such as alkaloids, flavonoids, terpenoids, polyphenols, and amino acids act as both reducing and capping agents, facilitating nanoparticle formation without the need for harmful intermediates (Jha et al. 2025). In biosynthesis, enzymes not only function as capping agents but also reduce electrons to produce a large number of nanoparticles with a very small size (Hayat et al. 2025).

ZnONPs have potent antimicrobial, antioxidant, and anti-inflammatory properties, making them ideal candidates for a wide range of biomedical applications such as drug delivery systems, wound healing, and therapeutic interventions (Ihsan et al. 2023). The capacity of zinc oxide (ZnO) to inhibit a wide range of harmful microorganisms, including fungi and both Gram-positive and Gram-negative bacteria, has made it recognized as a powerful antibacterial agent. Because of its small size, large surface area, and distinct surface reactivity, ZnO exhibits enhanced activity when it is in the form of nanoparticles (Hayat et al. 2025). The antibacterial activity of ZnONPs is mainly attributed to the generation of reactive oxygen species (ROS), which induce oxidative stress and cause severe damage to microbial cell membranes, proteins, and nucleic acids (Hayat et al. 2025). Further contributing to microbial cell death is the disruption of cellular metabolism and the compromising of membrane integrity caused by the release of Zn²⁺ ions (Sundrarajan et al. 2015).

Dysphania botrys is an annual herbaceous plant of the Amaranthaceae family. In folk medicine, D. botrys has been used to cure various diseases, including asthma, colds, influenza, headaches, liver and digestive disorders, and wound healing. It is also being investigated as a possible cancer treatment. D. botrys was chosen as a suitable plant for the preparation of ZnO nanoparticles through green synthesis due to their phytochemical characteristics and biological activities. This plant has a wide range of bioactive constituents that include flavonoids, phenolic acids, terpenoids, and essential oils (Dagni et al. 2022). These phytochemicals play an important role in both the reduction and stabilization of ZnO nanoparticles since they assist in transforming the metal precursors into ZnO nanoparticles while preventing the occurrence of agglomeration. Furthermore, due to the potent antioxidant capacity of D. botrys, the efficiency of the reaction is increased. Its antimicrobial properties could also contribute synergistically to the performance of the ZnO nanoparticles. The use of brine shrimp (Artemia salina) in a lethality bioassay is convenient because it is cost-effective and reliable, and results are available in a short time (Gangwar et al. 2024).

It is hypothesized here that D. botrys extract can mediate the green synthesis of ZnONPs and that these particles will show enhanced antimicrobial and antioxidant activities compared with the crude extract. This research paper highlights an environmentally friendly approach to producing zinc oxide nanoparticles using plant D. botrys for synthesis and stabilization. To our knowledge, there are no previous reports specifically describing D. botrys-mediated synthesis of ZnONPs; therefore, this work explores this plant as a new candidate for green ZnONPs production.

EXPERIMENTAL

Experimental Site

This study was conducted at the Institute of Biotechnology and Genetic Engineering, the University of Agriculture Peshawar, and PCSIR Laboratories.

Preparation of Extract

Fresh D. botrys plants were collected from the Medicinal Botanic Centre (PCSIR, Peshawar), washed with tap/distilled water, and shade-dried. The dried material was ground into a fine powder, and 15 g was mixed with 250 mL distilled water, heated at 100 °C for 1 h, and filtered (Whatman No. 1, 11 µm).

Preparation of Zinc Oxide Nanoparticles

The filtrate (pH 7.05) was combined with 0.1 M zinc acetate solution (2.195 g/100 mL), adjusted to pH 10 using NaOH, and stirred until the color changed from brown to yellow, indicating nanoparticle formation. The solution was centrifuged at 8,000 rpm for 25 min at 37 °C, washed thrice with distilled water, and the pellet was oven-dried at 65 °C for 24 h and ground into powder. Finally, the product was calcined at 400 °C for 1 h to obtain white ZnONPs, enhancing crystallinity and stability.

Physical Characterization of Zinc Oxide Nanoparticles

Characterization of ZnO nanoparticles (ZnONPs) was done using UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Absorbance measurements of ZnONPs solutions were taken by Cary 60 spectrophotometer (Agilent Technologies, Version 2.00, USA) for wavelengths ranging from 200 to 800 nm. For measurement, 1 mL of ZnONPs solution was prepared by dilution with distilled water using a quartz cuvette with a 1 cm path length and distilled water as the blank. XRD analysis was done using the Model 700 HS X-ray diffraction system (Shimadzu, Tokyo, Japan), which uses Cu Kα radiation with a wavelength of 1.5406 Å. The dried powder of zinc oxide nanoparticles was ground with an agate mortar and pestle and mounted on a glass holder. XRD pattern was obtained within the range of 10° to 80° 2θ angles.

FTIR spectra were obtained from plant extract as well as ZnONPs using a Bruker IR Biotyper device (Bruker Daltonics, Bremen, Germany). The ZnONPs and the freeze-dried plant extract powders were separately crushed with spectroscopic-grade potassium bromide (KBr) in the pellet form with the help of a hydraulic pellet press. FTIR spectra were taken between 4000 and 400 cm⁻¹. The SEM analysis of ZnONPs was performed using Hitachi TM1000 (52E-0101, Japan). A very little amount of dried ZnONPs was adhered on the carbon-coated aluminum stub by conductive double-sided carbon tape. The specimen was sputter-free (or gold-coated) and analyzed under high vacuum to investigate surface morphology and size of particles.

Phytochemical Analysis of the Plant Extracts via Gas Chromatography-Mass Spectrometry

GC-MS analysis was conducted on a methanol extract to characterize the major volatile and semi‑volatile phytochemicals of D. botrys. For plant extract preparation, 10 mL of methanol was added to the dried powder of D. botrys and kept at room temperature for 24 h to complete the extraction. The mixture was then filtered through a 0.45 μm membrane to remove residues, and the filtrate was concentrated using a rotary evaporator to obtain a solvent-free extract for GC-MS analysis. Phytochemical characterization was performed using a Shimadzu QP2010 Plus GC-MS system (Japan) following established protocols (Dagni et al. 2022). Operational parameters included a column oven temperature of 50 °C, an injection temperature of 250 °C, and spitless mode with a 1 µL injection volume. Separation was achieved using a 30 m × 0.25 µm capillary column, and 1 µL of the extract was analyzed for qualitative and quantitative identification of bioactive compounds.

Antimicrobial Activity

The antimicrobial activity of biosynthesized ZnONPs was assessed using the well diffusion method against five microorganisms: Escherichia coli ATCC 38738, Pseudomonas aeruginosa ATCC 39721, Staphylococcus aureus ATCC 36538, Bacillus subtilis, and Candida albicans. ZnONPs and D. botrys extract were prepared in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. After inoculating nutrient agar plates with microbial suspensions of 1 × 10⁶ CFU/mL, wells were created, and each well received 100 µL of either the ZnONPs suspension, D. botrys extract, or DMSO (negative control). The plates were incubated at 37 °C for 24 h, and the zones of inhibition were measured using a Vernier caliper. This study demonstrated the potential of ZnONPs and D. botrys extract as antimicrobial agents (Dagni et al. 2022).

Antioxidant Activity

The antioxidant activity of ZnONPs was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay (Alabri et al. 2014; Alawlaqi et al. 2023). A 0.1 mM DPPH solution was prepared by dissolving 3.94 mg of DPPH in 100 mL of DMSO and stored at 4 °C in the dark. For the assay, 1 mL of ZnONPs solutions (25, 50, 100, and 200 µg/mL in DMSO) was mixed with 1 mL of the DPPH solution, vortexed, and incubated in the dark at room temperature for 1.5 hours. Absorbance was then measured at 517 nm using a UV-visible spectrophotometer, with a blank sample and vitamin C (25 µg/mL of ascorbic acid) as a positive control. The percentage inhibition of DPPH radicals was calculated using the equation

Percentage of inhibition (%) = (A_control– A_sample)/(A_control) ×100 (1)

where A_control denotes the absorbance of the DPPH solution (without nanoparticles), and A_sample is the absorbance of the test sample (DPPH solution with nanoparticles).

Cytotoxic Activity

The cytotoxic potential of biosynthesized ZnONPs was evaluated using a brine shrimp lethality assay, based on the protocol by Younas et al. (2025). Artificial seawater was prepared by dissolving 38 g of synthetic sea salt in one liter of distilled water, filtering it through Whatman filter paper, and autoclaving it at 121 °C and 15 psi for 20 minutes. Approximately 1 g of Artemia salina cysts was hatched in the sterilized artificial seawater under constant illumination at 37 °C for 24 h to produce swimming nauplii. The cytotoxicity of the ZnONPs was assessed using serial dilutions of ZnONPs suspensions at concentrations of 200, 160, 120, 80, 40, 20, 10, and 2 µg/mL. In each test, a prepared ZnONPs suspension was added to vials containing two milliliters of artificial seawater and ten nauplii, bringing the total volume to five milliliters. After 24 h of incubation at 37 °C, the nauplii were checked for mortality, and the percentage of lethality was calculated according to Eq. 2.

Mortality (%) = (CN – ST) / CN × 100 (2)

where CN is the number of individuals in the control (or initial number), and ST denotes the number of surviving individuals after treatment.

Statistical Analysis

All experiments were performed in triplicate using three independent nanoparticle batches (biological replicates), and each measurement within an experiment was averaged from triplicate technical readings. The data were analyzed using SPSS statistical software (SAS Institute Inc., Cary, NC) with Duncan’s multiple range tests following analysis of variance (ANOVA).

RESULTS AND DISCUSSION

After the initial steps of plant collection and leaf grinding, followed by the addition of zinc acetate dihydrate and crystal formations, the plants were first subjected to GC-MS analysis to find their bioactive compounds or metabolites, which gave the plants their therapeutic potential.

GC-MS Analysis of Plant Extract

GC–MS analysis of the methanolic extract was conducted to identify major phytochemicals; however, as it differs from the aqueous extract used for nanoparticle synthesis, the detected compounds should be considered putative contributors to the synthesis process. The GC-MS analysis of the D. botrys extract revealed the presence of 26 bioactive compounds, with several compounds exhibiting notably high concentrations, which were significant for the study as shown in Table 1. Humulane-1,6-dien-3-ol was the most abundant compound, constituting 29.4% of the total concentration, followed by guaiol at 16.0% and spathulenol at 12.2%. Hinesol also showed a substantial presence at 10.6%, while 1,2,3,6-tetramethylbicyclo[2.2.2]octa-2,5-diene contributed 7.60%. Other compounds with significant concentrations included juniper camphor (5.06%), tridecanoic acid (3.39%), elemol (2.62%), and isolongifolene, 4,5-dehydro- (2.45%). Some of the phytochemicals present in D. botrys may participate in the reduction and stabilization processes during ZnONP synthesis. In some plant-mediated systems, enzymes and phytochemicals can act as reducing and capping agents. In the case of D. botrys, the focus was on total phytochemical content rather than identifying specific enzymes.

Table 1. The Bioactive Compounds Identified in D. botrys Extracted through GC-MS Analysis

These results are consistent with previous studies that found similar compounds in other plant species. For example, spathulenol was the predominant compound in Psidium guineense essential oil (80.7%) (do Nascimento et al. 2018). Hinesol was also one of the most abundant components (45.7%) in Atractylodes lancea (Ouyang et al. 2012), and 1,2,3,6-Tetramethylbicyclo[2.2.2]octa-2,5-diene was detected in Atractylodes macro-cephala and Astragalus membranaceus at 0.4% to 0.5% (Li et al. 2013). The oxygenated terpenoids detected using GC–MS (such as guaiol, spathulenol, hinesol, and elemol) that have hydroxyl groups function as reducing agents that reduce zinc ions (Zn²⁺) to zinc oxide (ZnO), while at the same time functioning as capping agents. This process entails reducing zinc ions to zinc hydroxide (Zn(OH)₂).

Analysis Through UV-visible Spectrometry

Dysphania botrys plant extracts were used to synthesize ZnONPs, with the initial verification confirmed by observing color changes and pH shifts upon the addition of zinc acetate dihydrate (Fig. 1A). The UV spectroscopy confirmed the formation of ZnONPs. The UV spectrum of ZnONPs synthesized from D. botrys extract is shown in Fig. 1A. The highest peak observed in the sample was at 365 nm, with an absorbance of 0.62, which demonstrates the presence of ZnONPs. The optimal absorption spectrum for ZnONPs typically lies within the range of 200 to 800 nm. The optical and structural properties of the biosynthesized ZnONPs exhibit significant variation depending on the type of plant material used, the types of ZnO precursors, and the protocol used. This aligns with previous reports: 375 nm for Plectranthus amboinicus (Vijayakumar et al. 2015), 375 nm for Aloe vera (Ali et al. 2016), and 365 nm for Salvadora persica (Al Rahbi et al. 2024).

X-Ray Diffraction for Crystalline Structure Analysis of ZnONPs

As depicted in Fig. 1B, the XRD pattern exhibited distinct diffraction peaks corresponding to the hexagonal wurtzite structure of ZnO, which aligned well with the standard reference (JCPDS card No. 00-001-1136). The prominent diffraction peaks were observed at 2θ values of 31.7°, 34.4°, 36.2°, 47.5°, 56.6°, 62.9°, 68.4°, and 77.1°, which are indexed to the (100), (002), (101), (102), (110), (103), (200), and (202) crystallographic planes, respectively. The sharpness and high intensity of the diffraction peaks indicate the excellent crystallinity of the synthesized ZnONPs. Furthermore, the absence of any additional peaks in the XRD pattern confirms the phase purity of the material, with no detectable secondary phases or impurities. The average crystallite size of the nanoparticles, calculated using the Debye-Scherrer formula, was 9.96 nm. These results collectively demonstrate the successful synthesis of highly crystalline and phase-pure ZnONPs. Similar findings have been reported for ZnONPs synthesized using Agathosma betulina extract (Thema et al. 2015) and Carica papaya leaf extract (Rathnasamy et al. 2017). According to the Debye–Scherrer equation, the average crystallite size of the prepared ZnONPs was calculated to be 9.96 nm. These findings are consistent with previously reported crystallite sizes of 17.47, 13.51, 10.34, and 9.04 nm for ZnONPs prepared from Camellia sinensis leaf extract at variable concentrations (Nava et al. 2017).

Fig. 1. UV-visible spectral analyses of biosynthesized ZnONPs showing the highest peak at 365 nm (A); and XRD graph showing Bragg reflection indicating the presence of biosynthesized ZnONPs (B)

Fourier Transform Infrared Spectroscopy

The FTIR analysis of both the plant extract and synthesized ZnONPs revealed a crucial interconnection, highlighting the dual role of phytochemicals in green synthesis as shown in Fig. 2. The presence of hydroxyl (O–H) and carbonyl (C=O) groups in both samples (at 3317 cm⁻¹ and 1632 cm⁻¹ in the extract, at 3287 cm⁻¹ and 1640 cm⁻¹ in ZnONPs) suggests that these functional groups act as reducing agents during nanoparticle formation and act as stabilizing agents through surface interactions. The shift in the O–H peak from 3317 cm⁻¹ to 3287 cm⁻¹ indicates partial consumption of hydroxyl groups during reduction, while the persistence of C=O peaks imply stabilization via chelation. The distinct Zn–O peak at 428 cm⁻¹ in ZnO spectra confirms successful nanoparticle synthesis. Overall, this analysis supports the role of plant-derived compounds in both reducing metal ions and stabilizing the resulting nanoparticles, aligning with green chemistry principles. Similar to the results observed for other green-synthesized ZnONPs, e.g., Zn–O peaks at 435 cm⁻¹ in Cayratia pedata biomass-mediated synthesis (Jayachandran et al. 2021) and 625.1 cm⁻¹ for Citrus medica-derived NPs (Sowmya et al. 2024).

Fig. 2. FTIR spectra showing the plant extract (A) and the biosynthesized ZnONPs (B)

Scanning Electron Microscope

The morphological characteristics of the green synthesized ZnONPs were analyzed using SEM, as shown in Fig. 3.

Fig. 3. Scanning electron microscopy (SEM) images of biosynthesized ZnO nanoparticles (ZnONPs) at different magnifications: (A) 0.5 µm scale bar and (B) 0.2 µm scale bar, showing the morphology and size distribution of the nanoparticles.

Based on the SEM analysis, the nanoparticles exhibited predominantly hexagonal particles, showcasing a highly uniform and consistent morphology. The distinct hexagonal structure of the ZnONPs highlights their crystalline nature and suggests a high degree of purity and precision in the synthesis process. These results agree with prior findings, where the ZnONPs synthesized from grains of Echinochloa frumentacea (Velsankar et al. 2020) displayed primarily a hexagonal morphology.

Energy Dispersive Spectroscopy of ZnONPs

The spectrum revealed the presence of primary elements, specifically zinc (Zn) and oxygen (O), which confirms the successful biosynthesis of ZnONPs. EDX spectra showed zinc and oxygen as the major elements, along with minor amounts of Mg, Si, and Ca, consistent with ZnO nanoparticles bearing trace inorganic impurities, as illustrated in Fig. 4. Previous studies have reported similar findings for the synthesis of ZnO from Myristica fragrans (Faisal et al. 2021) and from Ipomoea pescaprae (Venkateasan et al. 2017).

Fig. 4. EDX spectrum of green synthesized ZnONPs, which indicates different elements

Antibacterial Assay

The antibacterial activity of biosynthesized ZnONPs using D. botrys extract showed superior efficacy against both Gram-negative (E. coli, P. aeruginosa) and Gram-positive (S. aureus, B. subtilis) bacteria compared to the plant extract alone (Fig. 5). The plant extract (positive control) showed inhibition zones of 16±0.3 mm (E. coli), 15±0.1 mm (P. aeruginosa), 16±0.2 mm (S. aureus), and 14±0.1 mm (B. subtilis), while DMSO (negative control) had no inhibition. ZnONPs demonstrated significantly enhanced antibacterial activity, with inhibition zones of 18±0.2 mm (E. coli), 19±0.3 mm (P. aeruginosa), 22±0.3 mm (S. aureus), and 17±0.2 mm (B. subtilis), as shown in Fig. 5. When compared to standard antibiotics, ZnONPs exhibited competitive antimicrobial potential, particularly against S. aureus, where their inhibition zone (22±0.4 mm) was relatively close to that of ciprofloxacin (26±0.4 mm). These results highlight the promising role of D. botrys-mediated ZnONPs as eco-friendly antimicrobial agents. Their potency per unit mass was lower than that of standard antibiotics, and further optimization is needed. Similar results were reported for Myristica fragrans-derived ZnONPs against E. coli, P. aeruginosa, S. aureus, and B. subtilis (Faisal et al. 2021; Qanash et al. 2024) and ZnO from Ailanthus altissima fruit extracts (Awwad et al. 2020; Selim et al. 2025).

Fig. 5. Antibacterial activity against plant extract/ZnONPs: (A) S. aureus, (B) B. subtilis, (C) E. coli, (D) P. aeruginosa, (E) DMSO as a negative control, and the zone of inhibition of concentrations of ZnO (100 mg/mL), plant extract, and standard antibiotics (azithromycin and ciprofloxacin). The data represents the meaning of three replicates, with ±SE. Mean values in each bar followed by different lowercase letters are significantly different according to Duncan’s multiple range test at P < 0.05.

Antifungal Assay

Green-synthesized ZnONPs demonstrated promising antifungal activity against C. albicans, with an inhibition zone of 16±0.2 mm, surpassing the plant extract’s 14±0.1 mm, as shown in Fig. 6.

Fig. 6. Antifungal activity of plant extract (A), ZnONPs against C. albicans (B) and (C) zone inhibition (mm). Positive control of clotrimazole against C. albicans in replicates. The data represents the mean of three replicates, with ±SE. Mean values in each bar followed by different lowercase letters are significantly different according to Duncan’s multiple range test at P < 0.05.

This was less effective than clotrimazole (32.5±0.4 mm), which was used as a positive control. ZnONPs show potential as eco-friendly antifungal agents. Similar antifungal activity was also reported for ZnONPs synthesized from Salvia officinalis leaf extract against C. albicans (Yassin et al. 2023). The toxicity may involve oxidative stress and Zn²⁺ release, as reported elsewhere for ZnONPs, although we did not measure ROS or ion release in the present study.

Cytotoxic Assay

The cytotoxic potential of D. botrys-mediated ZnONPs was evaluated using a brine shrimp lethality assay over 24 h, revealing a strong concentration-dependent toxicity profile (Table 2). Notably, at 160 and 200 µg/mL, the ZnONPs induced 100% mortality within 24 h, signifying complete lethality. Lower concentrations also exhibited significant toxicity, with 80 and 40 µg/mL both causing 70% mortality, followed by 20 µg/mL (60%) and 10 µg/mL (50%). Remarkably, even at the lowest tested concentration (2 µg/mL), a persistent 50% mortality rate was observed after 24 h, highlighting the potent bioactivity of the synthesized nanoparticles. The IC50 value was notably low, indicating a strong cytotoxic effect, which is consistent with reports attributing ZnONP-induced oxidative stress and Zn²⁺ ion release as primary mechanisms of toxicity in Artemia salina. These findings highlight the potential of biologically synthesized ZnONPs as highly bioactive agents, warranting further exploration of their biomedical and environmental applications. Faisal et al. (2021) verify concentration-dependent toxicity on synthesized ZnONPs, possibly involving oxidative stress and bioaccumulation mechanisms. These findings highlight their dual potential in biomedical and environmental applications, despite their ecotoxicological risks.

Table 2. Cytotoxicity of ZnONPs on Brine Shrimp

Antioxidant Assay

The antioxidant potential of the green-synthesized ZnONPs was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay (Table 3). The results revealed a concentration-dependent increase in antioxidant activity, demonstrating the nanoparticles’ ability to scavenge free radicals effectively. At 25, 50, 100, and 200 µg/mL concentrations, the nanoparticles exhibited DPPH scavenging activities of 17.6%, 29.4%, 47.1%, and 58.8%, respectively. The corresponding absorbance values at 517 nm decreased progressively from 0.70 to 0.35, indicating a rise in radical scavenging efficiency with increasing nanoparticle concentration. Ascorbic acid (25 µg/mL), used as a standard antioxidant, showed 64.7% DPPH scavenging activity with an absorbance of 0.30, serving as a reference for comparison, as shown in Table 3. The results demonstrate the significant antioxidant potential of the biosynthesized ZnONPs, with their efficacy enhancing notably at higher concentrations. This suggests their promising role as an antioxidant agent in various applications. These results are in agreement with the previous reports of ZnONPs derived from sea lavender, which were dose-dependent (75.2% at 1000 µg/mL) (Naiel et al. 2022).

Table 3. DPPH Radical Scavenging Activity of ZnONPs

CONCLUSIONS

1. Many of the identified compounds, e.g., humulane-1,6-dien-3-ol (29.4%) and gauiol (16.0%), using GC-MS analysis, have been reported in the literature to possess antimicrobial, antioxidant, or anti-inflammatory activities, suggesting that D. botrys contains bioactive constituents that could contribute to the observed properties.
2. EDX elemental analysis confirmed high-purity ZnONPs composed mainly of zinc (72%) and oxygen (15%), with “ZnO-rich” nanoparticles with zinc and oxygen as predominant elements, accompanied by small amounts of other elements.
3. The biosynthesized ZnONPs exhibited strong antimicrobial activity, producing inhibition zones ranging from 16 to 22 mm against E. coli, P. aeruginosa, S. aureus, B. subtilis, and C. albicans.
4. Cytotoxicity assays demonstrated high toxicity toward brine shrimp larvae, indicating notable cytotoxic potential.
5. Antioxidant activity, assessed by the DPPH assay, showed a 58.5% free-radical scavenging efficiency, reflecting substantial antioxidant capacity.
6. Specifically, the findings provide partial support for the hypothesis formulated, because the biosynthesized ZnONPs showed a markedly higher antimicrobial and antioxidant activity than that of the crude extract, but the degree of increase differed for various microorganisms.
7. The D. botrys-mediated ZnONPs displayed in vitro antibacterial and antifungal activity and showed measurable antioxidant activity, along with notable toxicity in the brine shrimp assay. These preliminary findings indicate that D. botrys extract can serve as a green route to synthesize ZnONPs, but further work is required to optimize nanoparticle properties, fully elucidate mechanisms, and rigorously evaluate safety in mammalian and environmental models before any biomedical or environmental applications can be considered.

FUNDING

Supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies, and Scientific Research at King Faisal University, Saudi Arabia (Grant No. KFU260117).

ACKNOWLEDGMENTS

We thank the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. KFU260117), for supporting this research work.

Conflict of Interest

The authors declare no competing interests.

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Article submitted: January 7, 2026; Peer review completed: April 4, 2026; Revised version received and accepted: April 15, 2026; Published: April 24, 2026.

DOI: 10.15376/biores.21.2.5106-5121